LLLT预处理的BMSCs复合Si-CaP促进骨质疏松大鼠脊柱融合的实验研究

Osteoporosis has been considered as one of the worst diseases by WHO. Osteoporotic patients are not only at a higher risk of having bone fractures，but prone to develop spinal degenerative disease, trauma, infection, tumor, deformity of spine and other diseases as well. As for the treatment ,orthopedic surgeons usually apply spinal fusion procedure to reconstruct spinal stability. However, the incidence of spinal non-fusion and pseudarthrosis can go up to 30% in osteoporotic patients. It is a crucial task for orthopedic surgeons to seek a better way to treat osteoporosis and increase the success rate of spinal fusion. In recent years, with the development of tissue engineering technology and the enlightenment of Low Level Laser Therapy(LLLT), new ideas have been revealed to increase the possibility of spinal fusion in patients with osteoporosis. In this study, we will use low level laser(LLL) to irradiate the cultivative Wistar rats’ bone marrow stromal cells(BMSCs) which combined with the scaffold of silicate substituted calcium phosphate(Si-CaP) in vitro, then implant it into the spinal fusion animal model of osteoporotic Wistar rats, and irradiate the fusion site with LLL. Gross observation, manual palpation, X-ray, three-dimensional CT, Micro-CT, biomechanical test, molecular biology , bone histomorphometry and histological examination will be applied to investigate the outcome of spinal fusion. The improvement of bone structure, the differences of bone mass and the fate of the BMSCs in vivo will be observed too .The mechanism of LLLT will be explored with in vitro and in vivo studies . A new and effective method for the treatment of osteoporosis and the improvement of success rate of spinal fusion might be revealed.

骨质疏松症已被WHO列为十大最严重疾病之一。它不仅能导致骨折，还常合并脊柱退变性疾病以及脊柱的创伤、感染、肿瘤、畸形等疾病，治疗过程中，需通过脊柱融合术重建脊柱的稳定性。然而，骨质疏松患者脊柱不融合和假关节发生率高达30%。寻求既能治疗骨质疏松症，又能提高骨质疏松患者脊柱融合率的方法，成为骨科医生面临的重要课题。随着骨组织工程技术的发展和对低能量激光治疗(LLLT)的认识，为提高骨质疏松患者脊柱融合率提供了新的思路。本实验通过LLLT照射与硅磷酸钙(Si-CaP)支架载体复合培养的骨髓间充质干细胞(BMSCs)，并将预处理的BMSCs/Si-CaP植入骨质疏松大鼠脊柱融合的动物模型中，应用LLLT照射融合部位。通过力学、影像学、分子生物学、骨形态计量学、组织学等检测方法观察脊柱融合和骨质疏松的治疗效果，探讨LLLT的作用机制，为临床上治疗骨质疏松并解决其脊柱融合率低的问题提供新的有效方法。

目的：构建3D打印的Si-CaP支架载体，观察支架载体的表征及空间结构。将LLLT预处理的BMSCs与支架载体复合，观察BMSCs在支架载体上的生长情况，同时探讨该支架载体诱导BMSCs向成骨细胞分化的机制。将LLLT预处理的BMSCs/3D打印Si-CaP组织工程骨植入骨质疏松大鼠脊柱融合动物模型中，观察并探讨其促进脊柱融合的效果和成骨机制。方法：通过DLP技术制备3D打印的Si-CaP支架载体。应用扫描电镜和Micro-CT等方法，观察该支架载体的表征及空间结构。将LLLT预处理的BMSCs与构建的支架载体复合，扫描电镜和激光共聚焦显微镜观察BMSCs在支架载体上的形态和分布，并应用CM-Dil标记BMSCs；real-time PCR和Western blot测定各组BMP-2、β-catenin、p-β-catenin、ALP、OPN、OCN、COL-I、Runx2的表达情况。将LLLT预处理的BMSCs/3D打印的Si-CaP组织工程骨植入骨质疏松大鼠脊柱融合的模型中，通过大体观察、力学检测、影像学和组织学等方法，观察脊柱融合情况及BMSCs在体内转归情况，并观察LLLT治疗骨质疏松的情况。结果：构建的支架载体具有利于细胞粘附和增殖的表征和三维多孔立体结构。BMSCs在支架载体上分布较均匀，生长良好，增殖曲线明显；随着时间延长，各组成骨相关因子和信号通路蛋白表达逐渐升高。在LLLT和3D打印的Si-CaP支架载体双重作用下，BMSCs向成骨细胞分化的能力增强；通过大体观察、力学检测、影像学和组织学检测，可见构建的组织工程骨在骨质疏松大鼠脊柱融合过程中可提供良好的力学性能。随时间延长，组织工程骨逐渐降解吸收并发挥与自体骨相似的成骨效果。结论：3D打印的Si-CaP支架载体具有有利细胞粘附与增殖的表征和空间结构。在LLLT和3D打印的Si-CaP支架载体双重作用下可促进BMSCs向成骨细胞分化；构建的组织工程骨在脊柱融合的动物模型中，发挥了骨诱导、骨传导和骨生成的作用；移植的BMSCs参与脊柱融合的各个阶段并发挥成骨作用，LLLT预处理的BMSCs/3D打印Si-CaP组织工程骨通过Wnt/β-catenin信号通路可有效促进骨质疏松大鼠脊柱融合同时治疗大鼠骨质疏松。